The dear correlation between elevated levels of serum cholesterol and the development of coronary heart disease (CHD) has been repeatedly confirmed, based on epidemiological and longitudinal studies. The definition, however, of complex mechanisms of cholesterol transport in plasma, has allowed the recognition of a selective function of circulating lipoproteins in determining the risk for CHD.
There are, in fact, four major circulating lipoproteins: chylomicrons (CM), very low density (VLDL), low density (LDL) and high density (HDL) lipoproteins. Of these, HDL is directly involved in the removal of cholesterol from peripheral tissues, carrying it back either to the liver or to other lipoproteins, by a mechanism known as "reverse cholesterol transport" (RCT).
The "protective" role of HDL has been confirmed in a number of studies. Recent-studies directed to the protective mechanism(s) of HDL have been focused on apolipoprotein A-I (ApoA-I), the major component of HDL. High plasma levels of ApoA-I are associated with a reduced risk of CHD and presence of coronary lesions.
Plasma ApoA-I is a single polypeptide chain of 243 amino adds, whose primary sequence is known (Brewer et al. (1978) Biochem. Biophys. Res. Commun. 80: 623-630). ApoA-I is synthesized as a 267 amino acid precursor in the cell. The major structural requirement of the ApoA-I molecule is believed to be the presence of repeat units of 11 or 22 amino acids, presumed to exist in amphipathic helical conformation (Segrest et al. FEBS Lett. (1974) 38: 247-253). This structure allows for the main biological activities of ApoA-I, i.e. lipid binding and lecithin cholesterol acyl transferase (LCAT) activation.
The apolipoprotein A-IMilano (ApoA-IM) is the first described molecular variant of human ApoA-I (Francescini et al. (1980) J. Clin. Invest. 66: 892-900). It is characterized by the substitution of Arg 173 with Cys 173 (Weisgraber et al. (1983) J. Biol. Chem. 258: 2508-2513). The mutant apolipoprotein is transmitted as an autosomal dominant trait and 8 generations of carriers have been identified (Gualandri et al. (1984) Am. J. Hum. Genet. 37: 1083-1097). The status of a ApoA-IM carrier individual is characterized by a remarkable reduction in HDL-cholesterol level. In spite of this, the affected subjects do not apparently show any increased risk of arterial disease. Indeed, by examination of the genealogical tree it appears that these subjects may be "protected" from atherosclerosis.
The mechanism of the possible protective effect of ApoA-IM in the carriers seems to be linked to a modification in the structure of the mutant apolipoprotein, with the loss of one alpha-helix and an increased exposure of hydrophobic residues (Francheschini et al. (1985) J. Biol. Chem. 260: 1632-1635). The loss of the tight structure of the multiple alpha-helices leads to an increased flexibility of the molecule, which associates more readily with lipids, compared to normal ApoA-I.
Another very specific feature of the ApoA-IM, is its capacity to form dimers with itself and complexes with ApoA-II, in both cases because of the presence of the Cys residue.
To make possible production of sufficient quantities of ApoA-I in general, and more specifically ApoA-IM, use is made of recombinant DNA techniques, e.g in E. coli. Thus, recombinant preparation and use of ApoA-IM, monomers as well as dimers, are disclosed in patent specifications WO-A-88/03166 assigned to Farmitalia Carlo Erba (FICE), WO-A-90/12879 assigned to Sirtori et al, as well as WO-A-93/12143 and WO-A-94/13819 both assigned to Pharmacia AB (formerly Kabi Pharmacia AB).
Use of e.g. E. coli as medium introduces certain drawbacks. Thus, endotoxins or lipopolysaccharides (LPS) are high molecular complexes associated with the outer membrane (cell wall) of gram-negative bacteria, such as E. coli, Proteus and Salmonella. Endotoxins consist of two main parts, a lipid moiety called lipid A which is embedded in the outer membrane and a polysaccharide (O-antigen) which protrudes into the environment. Lipid A is the region which elicit the toxic effect of the endotoxins, a prerequisite being the presence of the entire lipid A moiety. The polysaccharide is made up of a O-specific chain and a core. The O-specific chain projects from the core and is the outermost part of the endotoxin. The core works as a linkage between lipid A and the O-specific chain.
It is known that endotoxins must be released from the bacterial surface to cause toxic effects. This happens when the bacteria multiply, at lysis and during stress. In aqueous solutions, free endotoxins form aggregates, micelles and vesicles, with a molecular weight of about 5 kDa up to &gt;10.sup.3 kDa.
It is known from the literature that several proteins form complexes with endotoxins. Particularly strong complexes are formed with HDL and apolipoproteins (Emancipator et al. (1992) Infect. Immun. 60: 596-601). According to Ulevitch et al. (1981) J. Clin. Invest. 67: 827-837, formation of a complex between HDL and endotoxins involve a two step mechanism, as follows: EQU Endotoxins.sub.(aggregated) .fwdarw.Endotoxins.sub.(disaggregated)(1) EQU Endotoxins.sub.(disaggregated) +HDL.fwdarw.Endotoxins-HDL (2)
This behavior has been confirmed e.g. by Munford et al (1981) Infect. Immun. 34: 835-843. There are indications suggesting that lipid A is the main factor in the complex and that the interaction involves both ionic and hydrophobic forces (Freudenberg et al. (1979) Nat. Toxins, Proc. Int. Symp. Anim., Plant Microb. Toxins., 6th, 349-354).
As already stated above, strong complexes are formed between endotoxins and HDL in general and particularly with apolipoproteins. This mechanism has been used in U.S. Pat. No. 5,128,318 assigned to the Rogosin Institute. U.S. Pat. No. 5,128,318 thus relates to HDL associated apolipoprotein containing reconstituted particles, and use thereof in removing lipid soluble materials, including endotoxins, from cells, body fluids, and the like. More particularly, U.S. Pat. No. 5,128,318 relates to a method for treating a subject for endotoxin-caused toxidty, by administering to the subject a reconstituted particle containing ApoA-I or ApoA-II, with or without cholesterol. Here, naturally, the aim is to create and maintain indefinitely the strongest possible complex, to avoid release of the endotoxins in the subject.
The complexes, strong in themselves, can be further strengthened e.g. by the presence of certain chemical compounds. Thus, deoxycholate is known to disaggregate endotoxins according to formula (1) above (Munford et al., see above and Emancipator et al., see above). The deoxycholate, then increases the binding of endotoxins to HDL according to formula (2). The result is a complex of endotoxins and HDL, which is very difficult to separate.
General methods for reducing or eliminating the effect of endotoxins are known previously. Thus, EP-A-494848 assigned to Pharmacia discloses methods for inhibiting endotoxin induced effects. A first embodiment relates to infusing a medicament containing arginine or arginine derivatives for treatment of an endotoxin induced effect, e.g. fever. A second embodiment relates to a method for removing endotoxins from water or aqueous solutions by filtering the water or aqueous solution through a bed containing immobilized arginine or an arginine derivative. To illustrate the second embodiment, tests were carried out with endotoxins from E. coli on columns containing Arginine SEPHAROSE.RTM. from Pharmacia Biotech of Uppsala, Sweden. The interaction is, however, weak and therefore much easier to separate into protein and endotoxins than would be the case with the strong complexes between apolipoproteins and endotoxins.
Anion-exchange chromatography is frequently used in the elimination of endotoxins from solutions containing proteins such as urokinase, interferon, asparaginase and albumin (Sharma (1986) Biotech. Applied Biochem. 8: 5-22). However, the interaction between the proteins and endotoxins is much weaker than the complexes formed between apolipoproteins and endotoxins.
EP-A-333474 to Mitsui Toatsu relates to a process for removing endotoxins from proteins by contacting an endotoxin-contaminated aqueous solution containing the protein with a protein adsorbent, washing the adsorbent with a solution containing an amino compound, and subsequently eluting the protein from the adsorbent. The only exemplified proteins are tissue plasminogen activator (t-PA), human serum albumin and inter-.alpha.-trypsin inhibitor. Examples of protein adsorbents are affinity, adsorption, hydrophobic and metal chelate chromatography gels.
Polymyxin B sulfate is an antibiotic polypeptide which has the ability to prevent the toxic effects of endotoxin by interaction with the lipid A moiety. Karplus and coworkers (Karplus et al. (1987) J. Immuno. Methods 105: 211-220) have used this knowledge for adsorbing endotoxins on Polymyxin SEPHAROSE.RTM. 4B, sold by Pharmacia AB of Uppsala, Sweden. Polymyxin is, however, in itself biologically active and therefore not suitable for removing endotoxin from solutions for intravenous injection (H. Matsumae et al. (1990) Biotechn. Biochem. 12: 129-140).
PROSEP.RTM. Remtox sold by Bioprocessing Ltd. of Great Britain, is a matrix prepared for specific removal of endotoxins from low and high molecular weight substances, such as antibiotics, vitamins, enzymes, antibodies and blood products. The gel consists of a low molecular weight, non-protein, non-carbohydrate synthetic ligand.
Charged filters are capable of removing endotoxins and other negatively charged molecules from different solutions. For example, Pall of United Kingdom offers POSIDYNE.TM. filters, consisting of a hydrophilic nylon 66 filter medium containing quaternary ammonium groups throughout the membrane structure. The retention capacity of the filter is independent of the temperature and is optimal at a pH of 5-8 and at a low flow rate.
There are presently several methods known for reducing or eliminating the influence of endotoxins in protein solutions generally. There is, however, no existing method to overcome the strong interaction between endotoxins and ApoA or ApoE. The present invention is intended to solve this problem.